The principal objective of the proposed research is to elucidate the mechanisms of regulation of nitric oxide (NO) production by NO synthase in mammalian cells. The field of NO research has virtually exploded in recent years and NO, one of the simplest molecules in nature, has developed into one of the most ubiquitous and multifaceted biological mediators in physiology and pathophysiology. In view of the widespread actions of endogenously generated NO as a vasodilator, inhibitor of platelet and leukocyte function, neurotransmitter in nonadrenergic-noncholinergic neurons, and pathophysiological mediator of target cell injury, the biosynthesis and metabolism of NO are critical processes that require understanding. NO is chemically labile in biological tissues and, therefore, the factors that influence the biological actions of NO will depend largely on the factors that affect the production of NO. The central hypothesis is that the biosynthesis of NO by vascular smooth muscle and endothelial cells, alveolar macrophages, and hepatocytes is tightly regulated by endogenous factors influencing both the enzymatic activity and transcriptional expression of NOS. Recent studies from this laboratory indicate that NOS activity can be altered by NO, nitroso compounds, tetrahydrobiopterin availability, and redox agents, and that compounds which interfere with the activation of nuclear factor kappa B (NF-kB) inhibit expression of inducible NOS in cytokine-activated macrophages. Additional experiments indicate that NO can down-regulate the transcription of mRNA for inducible NOS. Therefore, the proposed studies focus on the regulation of NOS activity and expression in mammalian cells. Five specific aims are proposed to achieve the objective: (1) to elucidate the mechanism by which NO inhibits neuronal and endothelial NOS; (2) to determine the reasons why macrophage inducible NOS is resistant to the inhibitory action of NO; (3) to elucidate the mechanism(s) by which calmodulin promotes the catalytic reaction of various isoforms of NOS; (4) to determine the conditions required for the production of reactive oxygen species by NOS isoforms; and (5) to elucidate the roles for NF-kB and reactive oxygen species as mediators of early response gene activation in the expression of inducible NOS in macrophages and hepatocytes. The proposed research represents a continuing long-term effort to understand the biological factors that influence the actions of NO in mammalian cells in both health and disease.